Chagas disease caused by the parasitic protozoan Trypanosoma cruzi remains a major public health concern in Latin America and is now spreading worldwide. At the chronic stage, the disease is commonly fatal. Indeed, serious cardiac and/or digestive symptoms develop in 20-40% of infected individuals 10 to 20 years later, with no curative therapies available. The progression to the chronic phase depends on parasite persistence in tissues. In this context, a series of molecular components of T. cruzi have been suggested as virulence factors which contribute to the severity of the disease. Oxidative assault to the parasite by exogenously- or endogenously-generated reactive species promoted by host cell-derived mediators has been revealed as a key mechanism accounting for parasite control. Indeed, superoxide (O2((), nitric oxide ((NO) and peroxynitrite (ONOO( ) appear to be molecular effectors for parasite cell death. However, the cellular origin (i.e. mammalian cell vs. T. cruzi), subcellular location and chemical characterization of the oxidizing species interacting with molecular targets in the parasite remain largely undefined. In turn, the parasites contain an array of enzyme-based antioxidant systems that attenuate or neutralize the effects of oxidants. Herein, we hypothesize that the redox balance provided by the T. cruzi antioxidant systems play a central role for virulence and parasite persistence in tissues and progression to the chronic phase. This hypothesis will be tested in infective and non-infective forms of T. cruzi, in infected cardiomyocytes and in a murine model of Chagas disease;the project also involves biochemical studies with purified antioxidant enzymes and the development and testing of specific redox-sensitive probes to study parasite oxidative stress. Three interrelated Specific Aims will be pursued: 1. Measure and characterize cardiomyocyte-induced T. cruzi oxidative stress with the use of novel methodologies and define its contribution to parasite control 2. Study the interactions of T. cruzi superoxide dismutases (TcFeSODs) with O2((-, (NO and ONOO( and examine their role in parasite programmed cell death 3. Assess the participation of the T. cruzi antioxidant network in the establishment of the infection and parasite persistence in vitro and in vivo. The research plan is designed to address at the molecular, cellular and animal levels relevant aspects in the pathogenesis of Chagas disease by assessing the contribution of the parasite antioxidant systems towards virulence and persistence. Successful completion of the proposed studies will 1) unambiguously establish the genesis of parasite oxidative stress during the infection process, 2) shed light on the contribution of the oxidant-antioxidant balance on parasite control, 3) determine the role of the parasite antioxidant network in disease severity and progression and 4) promote drug design and development. PUBLIC HEALTH RELEVANCE: The proposed studies will unravel how key components of the Trypanosoma cruzi enzyme antioxidant system participate in the susceptibility to and progression of Chagas disease. We anticipate identifying novel targets for disease prevention and treatment.